Synthetic oxygen transport made from cross-linked modified human or porcine haemoglobin with improved properties, method for a preparation thereof from purified material and use thereof

ABSTRACT

According to the claims, the present invention comprises the preparation of chemically modified, cross-linked hemoglobins with improved functional properties, the cross-linked hemoglobins prepared according to this method and the use of these hemoglobins as artificial oxygen carriers. The synthesis method is characterized by technical simplicity as well as by high yields. 
     Deoxygenated hemoglobin of high purity is conjugated covalently under the protection of an antioxidant with an effector of oxygen binding, especially with pyridoxal-5-phosphate, after which the hemoglobin is polymerized with glutardialdehyde as a bifunctional cross-linking agent. At the same time, there is a large increase in the volume of the reaction mixture and a decrease in the concentration of the hemoglobin during the addition of the cross-linking agent. Subsequently, after further dilution with water, a polyethylene oxide derivative is chemically linked to the cross-linked hemoglobins. Polymers are obtained, which are compatible with blood plasma and have optimized cooperativity and half saturation pressure values and can find use as artificial oxygen carriers and, in particular, are divided into a lower molecular weight fraction as blood substitute and a higher molecular weight fraction as blood additive, for example, for the treatment of oxygen-deficiency conditions.

This application is a 371 of PCT/EP01/06613, filed on Jun. 12, 2001.

In accordance with the claims, the present invention comprises theproduction of chemically modified, cross-linked hemoglobins withimproved functional properties, the cross-linked hemoglobins produced bythis method and their use as artificial oxygen carriers. The productionmethod is characterized by its technical simplicity as well as by highyields.

Deoxygenated hemoglobin of high purity is conjugated covalently underthe protection of an antioxidant with an effector for oxygen bonding,especially with pyridoxal-5-phosphate. After that, and the hemoglobin ispolymerized with glutardialdehyde with a very large increase in thevolume of the reaction mixture and, accordingly, a very great dilutionof the reactants during the addition of the cross-linking agent.Subsequently, after dilution with water, a polyethylene oxide derivativeis linked chemically to the cross-linked hemoglobins. Polymers withoptimized oxygen-binding characteristics are obtained, which arecompatible with blood plasma and, especially when divided into a lowmolecular weight and a high molecular weight fraction, can be used asartificial oxygen carriers as a blood substitute or blood additive, forexample, for the treatment of oxygen deficiency conditions.

For various clinical indications in medicine, it is desirable to haveavailable an artificial support system for the transport of oxygen. Inthe event of an acute loss of blood, it is not only appropriate toreplace the liquid volume isotonically and isocontically, but also torestore a further essential function of the blood, namely the transportof oxygen. With the decreasing preparedness to donate blood, adequatesupplies of blood are available less and less in the event of an acutecatastrophe or in the event of a war, particularly for covering anunforeseeable demand. The instantaneous availability of suitablesupplies of blood furthermore is associated with appreciable logisticproblems. Moreover, blood can usually be stored for only about 35 daysand must therefore be constantly replenished. This creates appreciablecosts. On the other hand, artificial solutions can be kept for asignificantly longer time, since they may optionally be frozen.Depending on the storage time, stored blood acidifies intracellularly.As a result, its acute oxygen binding characteristics are not by anymeans optimum and must be regenerated once again in the organism. On theother hand, an artificial oxygen carrier functions optimally from thefirst instant. The increasing lack of preparedness to donate blood iscontrasted, on the other hand, by the increase in demand of an agingpopulation. At the same time, because of the aging of the population,the number of potential blood donors has decreased. Likewise, because ofunpredictable risks of infection (immune weakness, hepatitis), thepopulation of the slums is unavailable as blood donors. An artificial,oxygen-transporting blood replacement would also be universal andindependent of the blood group. Moreover, it is possible that a volumedeficiency shock can be counteracted more rapidly with such a bloodreplacement than with stored blood, since the erythrocytes in the storedblood have stiffened and therefore have a lower capillary permeability.In any case, animal experiments have shown that a volume deficiencyshock can be combated more effectively with an oxygen-transporting bloodreplacement than with simple plasma expanders (Pabst, R. (1977): “OxygenTransport with Stroma-free Hemoglobin Solutions and Fluorocarbons”, Med.Klin. 72: 1555-1562, Keipert, P. E., Chang, T. M. S (1985):“Pyridoxylated Polyhemogloblin as a Red Cell Substitute forResuscitation of Lethal Hemorrhagic Shock in Conscious Rats: Biomater.,Med. Dev., Artif. Organs 13: 1-15). There are other applications for anartificial oxygen carrier, such as complicated surgical interventions,which necessarily are associated with high blood losses and can becarried out less and less, because of a lack of appropriate storedblood. On the other hand, larger and more invasive surgicalinterventions, including also transplants, are constantly beingdeveloped. In a particular case, the possibility of carrying out such anintervention depends decisively on the availability of a sufficientnumber of suitable stored bloods. Moreover, organs, which are to betransplanted, can be preserved far better, if they are perfused with(artificial) oxygen carriers. For instance, a liver transplant requiresup to 100 transfusion units of 450 mL each. In cases of polytraumaticinjuries, such as those caused by an automobile accident, similarlylarge amounts are required.

However, there is a need for an artificial oxygen-transporting, bloodadditive not only in the case of an acute loss of blood, but also in thecase of chronic blood-circulation disorders (especially cerebral,coronary, renal and peripheral—such as a sudden loss of hearing—or ananemic crisis, such as in the case of chronic osteomyelitis or aftertumor chemotherapy). The use of the additive is also an option in orderto prevent a threatening abortion in the case of a fetal oxygendeficiency due to a placenta insufficiency or to preventoxygen-deficiency injuries during birth. A requirement of this type iseven significantly greater than that in the aforementioned case of anacute blood loss. Such chronic circulation disorders are the cause ofdeath of about 750,000 people annually in Germany. In addition, thereare the cases of illness that arise from this cause. On the other hand,only about half that number of people die from cancer every year. It iswell known that attempts are made to treat chronic oxygen deficienciesin tissues by supplying hyperbaric oxygen. Aside from the fact that thistreatment is effective only as long as the oxygen overpressure existsand the procedure is not harmless, the danger of oxidative damage totissue due to reactions of free radicals of oxygen, which can bedetected by corresponding reaction products, exists here. An artificialoxygen carrier acts as long as it is present and offers so-calledlow-pressure oxygen to the tissue, so that the aforementioned damagedoes not occur.

The use of the oxygen-transporting blood additives as a temporarysupport for the endogenous oxygen transport system offers a furtherpossibility and alternative for combating a chronic tissue deficiency ofoxygen, the treatment of which was previously attempted withcirculation-promoting means, such as vessel dilators.

The fact that it is a functional oxygen treatment is very much to thebenefit of the concept. It is not the substrate (the oxygen) that isimproved; instead, the function of the carrier system, which brings theoxygen to the tissue is improved. This makes the treatmentmultiplicative in its effect and, with that, very effective and givesit, at the same time, a catalytic character. In addition, there areclear indications that an oxygen carrier, which is dissolved in plasma,is far more effective than one, which is “packed” in erythrocytes.

Moreover, such an artificial blood replacement can be produced free ofknown pathogens; infection problems, such as hepatitis and acquiredimmune deficiency (AIDS) are avoided in this manner.

An additional potential group of recipients for artificialoxygen-transporting solutions are patients, for whom an allergicreaction, for example, to HLA antigens, may be expected. Until now, theremoval of leukocytes from stored blood by filtration through cotton hasbeen attempted. On the other hand, artificial blood replacementsolutions would be completely free of leukocytes. Recently, it has beenobserved in pigs that, after an improvement in the oxygen supply(decrease in the oxygen affinity of the hemoglobin), the cardiac outputof the heart is decreased while the heart rate remains unaffected(Vilereal M. C., et al. (1987): “Engineered Red Blood Cells withModified Oxygen-Transport Properties: A New Oxygen Carrier”, Biomater.,Med. Dev., Artif. Organs 15: 397). In a different paper (Bosman, R. J.,et al. (1992): “Free Polymerized Hemoglobin Versus Hydroxyethyl Starchin Resuscitation of Hypovolemic Dogs”, Anesth. Analg. 75: 811-817”, itwas shown that the administration of oxygen-transporting solutions aftera volume deficiency shock in dogs prevented the increase in the cardiacoutput and accordingly is easy on the heart. By improving the oxygensupply, the possibility arises of achieving a functional cardiacprotection, which is helpful, for example, in the case of an infarction.This would be a completely new aspect of the use of oxygen-transportingsolutions.

A further use of such artificial oxygen carriers would be to increase inthe radiation sensitivity of tumors, especially since there areincreasing indications that molecular oxygen carriers, dissolved in theplasma, deliver the oxygen to the tissue far more effectively than doeswhole blood. Such artificial carriers bring about a synergism with thenative (intraerythrocytic) carrier. In other words, the molecularlydispersed artificial carrier in the blood plasma not only per se is bestat giving off oxygen from the capillary, but also intensifies the oxygendelivery of the existing native system by way of the mechanism offacilitated diffusion. This means that only a very low concentration ofthe artificial carrier in the plasma is required for this purpose.Nevertheless, this functional therapy (see above) remains extremelyeffective.

Everything, which has been stated, makes clear the need for anartificial oxygen transporter. It is indispensable for the use of anartificial oxygen carrier that its starting material is available insufficient amounts. Deteriorated stored blood accordingly does not solvethe problem. It is therefore necessary to use animal hemoglobins,preferably from the most important slaughtered animals, namely cattleand/or pigs.

Roughly two types of applications arise from the presentation of theneed for artificial oxygen carriers, on the one hand, in the case of aheavy loss of blood and, on the other, in the case of a chronic oxygendeficiency. In the first case, an iso-oncotic, oxygen transportingvolume substitute (artificial oxygen carrier of the first generation) isrequired as compensation and on the other hand, in the second case, anoxygen-transporting blood additive (artificial oxygen carrier of asecond and new generation) is required. As already mentioned, the lattercase is far more frequent. Moreover, an appropriate blood additive, incombination with so-called plasma expander, also permits an acute lossof blood to be treated with the great advantage that the physician hasthe possibility of coordinating the administration of oxygen carriers aswell as the liquid volume with the requirements with the individualpatient.

The organism is also able to change both (the amount of oxygen carrierand the blood volume) independently of one another, namely theerythrocyte formation by way of the erythropoetin and the plasma volumeby way of its own regulation system. The two parameters are uncoupledowing to the fact that the carrier has a much smaller colloidal osmoticpressure than does the plasma.

Previously, three basically different strategies for developing anartificial oxygen carrier for blood were employed by others (state ofthe art: Rudolph, A. S. et al. (Publisher) Red Blood Cell Substitutes:Basic Principles and Clinical Applications, Marcel Dekker, New York, etal., 1998; Tsuchida E. (Publisher): Blood Substitutes: Present andFuture Perspectives, Elsevier Science, Amsterdam, 1998; Chang, T. M. S.(Author & Publisher) Blood Substitutes: Principles, Methods, Productsand Clinical Trials, Volume 1 and Volume 2, Karger Landes, Basel et al.1997 and 1998).

The use of emulsions with fluorinated hydrocarbons—recently, otherhalogens, such as bromine, have also been used—in which oxygen isparticularly soluble (Hirlinger, W. K., et al. (1982): Effects of aPartial Blood Exchange with Fluosol DA 20% on the intact organism of thepig”, Anästhesist 31 660-666). However, since the fluorinatedhydrocarbons are lipophilic, it is to be expected that interactions anddisorders in the lipid layers of the cell membrane will occur. The laterare integrating, functional components of the cell. Moreover, thefluorinated hydrocarbons must be disposed with emulsifiers, such asphospholipids, which can interfere additionally with the membranes ofthe cells (so-called artificial oxygen carriers, based on fluorinatedhydrocarbons of the first generation).

A further strategy is represented by the microencapsulation of highlyconcentrated solutions of natural and also of chemically modifiedhemoglobins in phospholipid vesicles with addition of suitable effectorsof hemoglobin binding (“artificial erythrocytes or hemosomes”) (Ogata Y.(1994): “Characteristics of Neo Red Cells, Their Function and Safety:In-Vivo Studies”, Artificial Cells, Blood Substitutes, andImmobilization Biotechnologies 22: 875-881). The first animalexperiments in this field have been successful (Hunt, C. A. et al.(1985): “Synthesis and Evaluation of a Protypal Artificial Red Cell”,Science 230: 1165-1168). The vesicles had a diameter of less than 0.05μm and their volume therefore was more than two powers of ten smallerthan that of natural red blood cells.

The third strategy consists of the production of infusible hemoglobinsolutions. The artificial oxygen carrier then occurs extracellularly inthe blood. The first two solutions of the problem can be regarded asrelating to the production of an artificial blood, so that thedifficulty of a colloidal, osmotic interference cannot occur. On theother hand, for this solution of the problem, there was a plasmaexpander at the very start, the macromolecules of which can alsotransport oxygen (a so-called, artificial oxygen carrier), on the basisof hemoglobin of the first generation.

Native hemoglobin cannot be used for this purpose, for example, becauseit is eliminated too rapidly by way of the kidneys. A chemicalmodification therefore is indispensable. For example, within the scopeof this strategy, hemoglobin has been bound by its amino groupscovalently to dextrans or has itself been polymerized to a molecularweight of 700,000 g/mole. The former procedure was pursued, for example,by Firma Fresenius (Fresenium E. (1976), Blood and PlasmaSubstitute—Comprising a Colloidal Solution of Hydroxyethyl StarchCoupled to Hemoglobin-Free Stroma”, Patent DE-P 2616-086) and the laterby Firma Biotest (Bonhard K., et al. (1983): “Method for ObtainingHepatitis-Safe, Sterile, Pyrogen-Free and Stroma-Free HemoglobinSolutions”, Patent DE-O 31 30 770) and Firma Alza (Bonsen P. (1976):“Water-soluble Polymerized Hemoglobin”, Patent DE-O 26 07 706). Afurther strategy with regard to extracellular solutions is thestabilization of hemoglobin by cross-linking it to an intratetramer orby appending side groups (oligo ethylene glycol) without significantlyincreasing the molecular weight of the tetramer (stabilized hemoglobins(Matsushita M. et al. (1987): “In Vivo Evaluation of PyridoxylatedHemoglobin-Polyoxythene Conjugate”, Biomat, Artif. Cells. Artif. Org.15: 377). As mentioned above, extracellular blood replacement solutionshave given promising results with respect to treatment of shock inanimal experiments.

The use of hemoglobin as an artificial oxygen carrier has an advantageover the use of fluorinated hydrocarbons, because the advantageousproperties of the natural binding of oxygen can be utilized. Theseinclude the optimally adapted oxygen affinity, the homotropiccooperativity, that is, the S-shape of the oxygen binding curve, as wellas the (alkaline) drilling effect which forms the basis of a natural,self-regulatory mechanism for the selective delivery of oxygen todeficiently supplied tissue.

It is clearly evident from the relevant literature that anintra-tetrameric, covalent bonding of hemoglobin units (Keipert P. E.,et al. (1989): “Metabolism, Distribution, and Excretion of HbXL: ANondissociation Interdimerically Crosslinked Hemoglobin with ExceptionalOxygen Offloading Capability”,—in: Change, T. M. S., Geyer R. P. (Eds.):Blood Substitutes, Marcel Dekker, New York 1989) and/or a polymerizationof the hemoglobin leads to a large increase in the residence time in theblood. (Chang T. M. S. (1987): “Modified Hemoglobin as Red Cell BloodSubstitutes”, Biomater., Med. Devices Artif. Organs 14: 323-328;Friedman H. J., et al. (1984): “In Vivo Evaluation ofPyridoxylated-Polymerized Hemoglobin Solution”, Surg. Gynecol., Obstet.:159 429-435). This is an essential perquisite for the clinicalusefulness of such solutions.

However, in the case of extracellular, molecularly disposed artificialoxygen carriers, a very large area of need, the chronic oxygendeficiency, has been ignored by aiming for iso-oncotic solutions. Asalready mentioned, the significantly more frequently occurringconsequences of chronic circulation disorders can, however, be improvedonly by oxygen-transporting solutions, the colloidal osmotic pressure ofwhich can be disregarded with respect to the normal (35 mbar), that is,only with the help of an oxygen-transporting blood additive, a“molecular erythrocytes concentrate”, as it were. These are artificialoxygen carriers based on hemoglobin of a second generation.

The following problems arose during the different attempts to develop anartificial oxygen transporter:

-   Increase in the hemoglobin affinity for oxygen: due to the chemical    modification of the hemoglobin molecule, the half saturation    pressure (P50) is decreased. As a result, the delivery of oxygen to    the tissue is made more difficult. This occurs in a pronounced    manner during the binding of hemoglobin to dextran. In order to    avoid the increase in oxygen affinity, suitable effectors (such as    pyridoxal phosphate) have been linked to the prosthetic group of the    hemoglobin.-   Frequently, the so-called n50 value (HILL index) as an expression of    the decreased homotropic cooperativity (weakened S shape of the    oxygen-hemoglobin binding curve), is decreased at the same time;    this also makes it more difficult to supply the tissue with oxygen.    This S shape of the oxygen-hemoglobin binding curve at the same time    facilitates the absorption of oxygen in the lung and its delivery to    the cells. On the other hand, fluorinated hydrocarbons have a linear    “oxygen binding curve” and therefore do not have this functional    advantage.-   The artificial oxygen carrier frequently has too short a residence    time in the organism, the dissolved hemoglobins being eliminated by    way of the kidneys. In the case of extracellular hemoglobin    solutions as artificial oxygen carriers, the attempt has been made    to prevent the elimination by intermolecular cross-linking;    nevertheless, the residence time of the extracellular hemoglobins    remain shorter than desired. On the other hand, hemosomes are    removed from the plasma by the reticuloendothelial system of the    organism. For example, the half life of the artificial erythrocytes    (see above) was 5.8 hours.-   Excessive colloidal osmotic pressure: This can result in a loss of    volume (volume deficiency shock). This effect occurs when the    molecular weight of the artificial oxygen carrier is comparable to    that of the plasma proteins. As a result, the dosage of the    artificial oxygen carrier cannot be chosen freely and, instead, the    oncotic relationships must be taken into consideration.-   The oncotic milieu of the plasma continues to be determined    primarily by the so-called second virial coefficient (A₂ value).    This characterizes the interaction of the (always macromolecular)    oxygen carrier with the solvent (water). The synthesis should be    conducted so that this value is close to zero.-   Excessive viscosity of the carrier solution: Usually, this is    associated with an A₂ value, which is too large, and it occurs    preferably when a carrier consists of chain molecules. According to    the Einstein viscosity law, an excessive viscosity does not occur if    the polymeric carrier molecules are spherical and compact.-   In vitro stability of the carrier molecules: This refers, on the one    hand, to the decomposition of the molecules and, on the other, to    the oxidative binding of methemoglobin, which is no longer capable    of binding oxygen, and finally to the viscosity due to the slowly    changing interactions between the carrier and the albumin of the    plasma.-   Excessive reaction of the reticuloendothelial system (RES): The main    influencing factor is the molecular size of the artificial carrier.    The critical limit for this is about 0.3 μm. Larger particles    activate the RES.-   Kidney and liver damage: a kidney shock occurs especially if the    stroma-containing hemoglobin solution is used. Ever since the    solutions were subjected to ultrafiltration, kidney damage has no    longer being observed. Liver damage was indicated with the help of    the plasma transaminase mirror; it is presumably based on cellular    membrane interactions: the liver has an open flow path (fenestrated    capillaries).-   Hemostasis must also be checked: disorders in the sense of    preventing and inhibiting hemostasis are conceivable; special    attention must be paid to thrombocyte aggregation.-   Antigenic effects: In this connection, it was recently shown in    homolog experiments in rats that native hemoglobin has no antigenic    activity and that the polymerization with glutardialehyde has not    increased the antigenicity (Hertzman, C. M., et al. (1986): “Serum    Antibody Titers in Rats Receiving Repeated Small Subcutaneous    Injections of Hemoglobin or Polyhemoglobin: A Preliminary Report”,    Int. J. Artif. Organs 9: 179-182). In the same paper it is shown    that native and polymerized human hemoglobin has little antigenic    activity in rats and that the effect is intensified at most slightly    by polymerization.-   Toxic effects can be differentiated as pyrogenic,    vasoconstrictive—for example, on coronary vessels—and endotoxic. The    vasoconstrictive effect is based presumably on entrapping nitrogen    monoxide radicals, which are, and is well known, endogenous,    vasodilatoric control substances; however, the vasoconstrictive    effect can also be explained by the high effectiveness of the    artificial carrier, in that the smooth musculature is supplied “too    well” with oxygen.-   In vivo stability: An endogenous, enzymatic degradation, for    example, by proteases, must be considered.-   Overloading the organism with lipoids (emulsifiers): this    complication occurs only when microencapsulated hemoglobin solutions    of fluorinated hydrocarbons are used; a complement activation by way    of the alternative path as well as antibody formation was noted    here.-   Compatibility of the artificial carriers with fresh, human blood    plasma, there may be precipitation, depending on the pH.

Presumably, because of the problems listed here, an artificial oxygencarrier is as yet not available for routine clinical applications. It isevident from the various problems named that a usable artificial oxygencarrier must still satisfy a larger range of requirements.

In nature, oxygen is always transported in microscopic giant aggregates.Two basically different “problem solutions” have been developed here. Inthe case of higher animals (and also in man), the molecular oxygencarrier is packed in cells (in the erythrocytes). Secondly,oxygen-binding giant molecules develop, which are dissolvedextracellularly in a hemolymph and not intracellularly. This variationis encountered predominantly in lower elements to different degrees.Annelides, for example, have high molecular weight hemoglobins,averaging about three million g/mole, as oxygen carrier. Furthermore,there are so-called hemerythins, for which the iron, as oxygen binder,is released molecularly directly with the protein. Finally, there arethe hemocyanins with a molecular weight of about eight million g/mole,for which copper is the oxygen-binding heavy metal ion (see alsoBarnikol, W. K. R., et al. (1996): “Hyperpolymeric Hemoglobins andArtificial Oxygen Carriers. An Innovative Attempt at MedicalDevelopment”, Therapiewoche 46: 811-815). The historical transition fromextracellular dissolved oxygen carriers to the erythrocytes has causedan increase in the oxygen content of the blood fluid by a factor ofthree. For example, 1 mL of the “blood” of the earthworm can bind atmost 3.6 μmoles of oxygen. On the other hand, up to 9.0 μmoles of oxygenare bound in the same volume of human blood.

The earthworm has giant molecules (erythrocruorin) in its blood asoxygen carrier with a molecular weight of about 3,400,000 g/moles andapproximately 200 binding sites for oxygen. Moreover, the molecule isvery compact and its quaternary structure is highly ordered. Themolecule is so large, that it can be made visible directly with the helpof the electron microscope. Its concentration in the hemolymph is atleast 6 g/dL. Measuring the oxygen-binding curve under simulated in vivoconditions gives a half saturation pressure (P50) of 9.1 torr (Barnikol,W. K. R. and O. Burkard (1987): “Highly Polymerized Human Hemoglobin asan Oxygen-Carrying Blood Substitute”, Advances in Experimental Medicineand Biology, vol. 215: 129-134; Barnikol, W. K. R. (1986) “The Influenceof Glutardialdehyde on the Oxygen Cooperativity of Human Hemoglobin”,Pflügers Archiv 406: R 61). This system thus supplies the cells of theearthworm adequately with oxygen. Due to the extremely high molecularweight, the hemoglobin of the earthworm has practically no colloidalosmotic effect anymore (only about 0.4 mbar). With that, as in the bloodof mammals—the oncotic pressure of the erythrocytes is only 10⁻⁷torr—the two functions of colloid osmolarity and oxygen binding areuncoupled and both can be varied freely as control variables of theorganism.

If the principle of the earthworm system is to be transferred toartificial oxygen carriers, which are based on human or animalhemoglobin, the hemoglobin molecule must be changed so that, asextracellular giant molecule with a negligible oncotic pressure, it cansupport the normal oxygen transport of the erythrocyte at leastintermittently. Such artificial oxygen carriers then are highlypolymerized hemoglobins, which must take into account all the problemsmentioned above. Earthworm “hemoglobin”, which admittedly fulfills theoncotic pressure requirement, will not be usable for man for thispurpose, because it presumably has too high an antigenicity; moreover,the half-saturation pressure of 9 torr is too low (Barnikol, W. K. R andO. Burkard (1987): “Highly Polymerized Human Hemoglobin as anOxygen-Carrying Blood Substitute”, Advances in Experimental Medicine andBiology, vol. 215: 129-134; Barnikol, W. K. R. (1986): “The Influence ofGlutardialdyde on the Oxygen Cooperativity of Human Hemoglobin”,Pflügers Archiv 406: R 61). Moreover, it can presumably not be obtainedin the amounts required.

Until now, in the development of artificial oxygen carriers, attemptswere made to adjust the half saturation pressure of the carriersprecisely to the normal value of about 20 torr in man. However, animalexperiments showed that a molecularly disperse artificial oxygen carrierwith a half saturation pressure of about 15 torr oxygenates organs best(Conover et al. (1999), Art. Cells, Blood Subst. Immobil. Biotech. 27:93-107). On the other hand, however, animal experiments also show that asufficiently large oxygen capacity of the blood is at least equallyimportant for an adequate oxygen supply (Moss, G. S., et al (1984):“Hemoglobin Solution—From Tetramer Polymers”, In: The Red Cell: SixthAven. Arbor Conference, Alan R. Riss, N.Y., 1984: 191-210). In turn,this depends on the possible concentration of the oxygen carrier in theplasma. Furthermore, it was shown here that it is not necessary toadhere to a half saturation pressure of 26 torr. Rather, it is essentialthat a certain critical value must be exceeded.

It is a further requirement of the development of an artificial oxygencarrier that a manufacturing process be as simple as possible and, withthat, economic, especially since it is necessary to work under sterileconditions from the start. The yield of the product should be high andmaterial for use as blood additive and material for use asoxygen-transporting blood-volume substitutes should be formedsimultaneously during the cross-linking reaction.

Furthermore, during the production of the artificial polymers fromhemoglobin by linking the hemoglobin molecules over their amino groupsby means of suitable, bifunctional cross-linking agents, particularattention must be paid to the fact that molecular networks, which areinsoluble and therefore decrease the yield, are not formed. For thisreason, the formation of the so-called percolation distribution of themolecular weight is to be prevented.

It is therefore an object of the present invention to be able to producehypo-oncotic, artificial oxygen carriers from cross-linked hemoglobins,which have optimized, good functional properties, especially thecharacteristics of oxygen binding, and are suitable for use aspharmaceutical products in man, in a technically simple process in alarge yield.

Pursuant to the invention, this objective is accomplished as describedbelow. Surprisingly, it was possible eliminate the fundamental problemof the formation of a percolation distribution of the multimerizationdegrees and molecular weights by the cross-linking of the polyfunctionalhemoglobins with the bifunctional cross-linking agent, glutardialdehyde,by greatly increasing the volume of the reaction mixture (2- to 10-foldin all) during the cross-linking, and, moreover, adding initially thecross-linker as a dilute solution and subsequently additionally dilutingwith water. By these means, cross-linked hemoglobins with a high degreeof cross-linking are formed in a large yield, without developing apercolation distribution of the molecular weight and forming insoluble,molecular networks. Rather, the crude product of the cross-linking isdistinguished by a certain, desirable (approximate) upper limit of themolecular weight range. A so-called quadrilateral distribution of themolecular weight is obtained in the gel chromatogram (see FIGS. 1, 2 and3).

The inventive preparation thus comprises the following steps:

In a one-vessel reaction, hemoglobin

-   i) initially is deoxygenated, especially by passing nitrogen over    it,-   ii) subsequently reacted covalently with a chemically reactive    effector of the oxygen binding,-   iii) after which the solution is treated with a chemically    unreactive effector and then-   iv) the hemogloblin is cross-linked stably and covalently with    glutardialdehyde with a very great dilution (2- to 5-fold of the    volume of the reaction mixture, with simultaneous addition of the    cross-linker (in solution)) and subsequently the reaction volume is    increased further with water, the total dilution of the solution    being 2- to 10-fold, especially 2- to 7-fold and particularly 5- to    6-fold and then-   v) a polyethylene oxide is linked covalently.

The product obtained can be worked up by known methods.

Preferably, the starting hemoglobin originates from pigs or from man,pigs and especially domestic pigs being particularly preferred.

Particularly and pursuant to the invention, the glutardialdehyde isadded in step iii) in a very highly diluted solution in atime-controlled manner. Preferably, there is a subsequent furtherdilution with water and an increase in the reaction volume, so that theabove-mentioned total dilution is achieved.

Moreover, in step iv), glutardialdehyde is preferably added in a molaramount of 6 to 10 and especially of 7 to 9 moles/mole, based on themonomeric hemoglobin, dissolved in 1-4, preferably 1-2, particularly1.5-2 and especially 1.7-1.9 L of water per liter of original reactionsolution.

This addition is timed to take place between about 3 and 15, especiallybetween 3 and 10 and particularly between 4 and 6 minutes. Subsequently,the solution is reacted for 1 to 6 hours.

It is furthermore preferred that, before the reaction of step ii), 2-8,especially 3-6 and particularly 3-4 moles of sodium ascorbate per moleof uncrosslinked hemoglobin be added to the solution containing thehemoglobin. This reaction takes place over a period of 0.5 to 6 hoursand especially of 70 to 120 minutes.

Furthermore, in step ii), preferably pyridoxal-5′-phosphate is linked aseffector in a molar ratio, based on the monomeric hemoglobin, of 0.5 to3, preferably of 1 to 2.5 moles/mole covalently over a period of 0.5 to20 and especially of 1 to 7 hours.

Pursuant to the invention, and furthermore preferably andadvantageously, reductive sodium borohydride is added in step ii) aswell as in step iv) after the covalent linking of pyridoxal-5′-phosphateand of glutardialdehyde.

This is added particularly in step ii) in an amount of 1 to 9,preferably of 1 to 5 and particularly of 1 to 2.5 moles/mole, forexample, over a period of 30 to 90 minutes, and in step iv) in an amountof 5 to 20 and especially of 6 to 12 moles/mole, in each case based onthe monomeric hemoglobin, over a period of 15 to 100 minutes.

It is particularly preferred if, as unreactive effector in step iii),2,3-bisphosphoglycerate is added in an amount of 0.5 to 6 and especiallyof 1 to 4 moles/mole, based on the monomeric hemoglobin, and if about 5to 50 minutes, especially 10 to 20 minutes and particularly 15 minutesthereafter glutardialdehyde is added.

As polyethylene oxide, preferably a polyethylene glycol ether with, forexample, a C1-C5 alkyl group, such as methyl, ethyl and butyl, with amolecular weight of 500 to 3000 g/mole, especially amethoxy-polyethylene glycol derivative with a molecular weight of 1500to 2500 g/mole and especially of 2000 g/mole, such as, especially,methoxypolyethylene glycol succinimidyl propionate, in amounts of 2 to12 and especially 3 to 8 moles/mole of hemoglobin. Other derivatizingproducts are methoxy-polyethylene glycol succinimidyl succinamide andmethoxy-polyethylene glycol succinimidyl hydroxyacetate. The linking ofpolyalkylene oxide to uncrosslinked hemoglobin is described in U.S. Pat.Nos. 4,179,637, 5,478,805 and 5,386,014 and the EP-A 0 206 448, EP-A 0067 029 and the DE OS 3,026,398.

The reaction is carried preferably over 1 to 4 and particularly over 2to 3 hours.

It is furthermore advantageous that all synthesis reactions take place,in particular, in solutions freed from oxygen by tonometry withoxygen-free gases. The method used is described by H. Potzschke in“Hyperpolymers of Human Hemoglobin, Development of Preparative Methodsfor their Synthesis, Validation of Analytical Methods and Equipment fortheir Characterization”, Dissertation, Medical Faculty, University ofVienna, 1997.

The product obtained can be worked up in the usual manner, as describedin the following. In particular, it has a molecular weight distributionof 50,000 to 5,000,000 and optionally up to 10,000,000 g/moles or more.

Preferably, the product obtained can be divided into a fraction of highaverage molecular weight and a fraction of low average molecular weight,the boundary preferably being at 700,000 g/moles, by a preparative,material-separating method, such as a preparative volume exclusionchromatography, an ultrafiltration, a fractional precipitation, forexample, with polyalkylene oxide or salt such as ammonium sulfate asprecipitant, or a field-flow fractionation method (Curling, J. M(publisher), “Methods of Plasma Protein Fractionation”, Academic Press,London et al. 1980, as well as the patents EP-A 0 854 151 and EP-A 95107 280).

A pharmaceutical preparation can be produced from the product of highmolecular weight and also from the product of low molecular weight, aparenteral blood substitute being obtained from the low molecularfraction of the polymers and a parenteral blood additive from the highmolecular weight fraction of the polymers.

Before (and after) the individual steps of the reaction, the pH isadjusted preferably with lactic acid or sodium hydroxide solution tovalues between 6 and 10, depending on the reaction step, for example, toa value of 6.5 to 7.5 before step ii), subsequently to 7.5 to 8.5 andsubsequently again to 6.5 to 7.5 before step iii), after that to 7.5 to9, as well as to 7.5 to 10 before step v).

The concentration of hemoglobin preferably is 200 to 380 g/L andparticularly 240 to 360 g/L; the solution furthermore contains 10 to 150mmoles/L of sodium bicarbonate and 10 to 150 mmoles/L of sodiumchloride.

During the “one-vessel reaction”, the temperature is 2° to 42° C.,especially 3° to 25° C. and particularly 4° to 24° C.

The artificial oxygen carrier, produced pursuant to the invention,preferably has an n50 value (cooperativity) of 1.6 to 2.5 and a p50value (semi-saturation pressure) of 16 to 24 torr.

The product, which is obtained pursuant to the invention and has thecharacteristics given, can be used to produce an agent for an intravasalor biomedical application as an artificial oxygen carrier, or in theform of a pharmaceutical preparation as a blood substitute or as anadditive to blood to increase the oxygen transport capacity (bloodadditive) or to a nutrient solution in human and animal organisms, inindividual organs or in biotechnical applications, especially for thetreatment of a chronic oxygen deficiency in man.

To prepare the products, which are to be administered, the inventivehemoglobin derivatives are dissolved in suitable media, such as infusionsolutions, for example in aqueous salt or glucose solutions, preferablyin concentrations isotonic with the blood plasma.

The inventive method is based accordingly on individual reaction steps,which are matched to one another and the significance and effects ofwhich are explained in the following.

The starting material is a very pure hemoglobin. This may be obtained byknown methods from the fresh blood of slaughtered animals or, forexample, overaged stored blood. Methods for obtaining purifiedhemoglobins are described by Antonini, E. et al. (publisher) in:“Hemoglobin”? (Colowick, S. P. and N. O. Kaplan (publishers): Methods inEnzymology, Vol. 76) Academic Press, New York et al. 1981.

As mentioned, pursuant to the invention, during the cross-linking withglutardialdehyde, which is known (DE 24 99 885, U.S. Pat. Nos.4,857,636, 4,001,200, 4,001,401 and DE 449 885, U.S. Pat. No. 5,439,882,EP A 0 221 618), the hemoglobin is deoxygenated (that is, freed from itsphysiological ligands, oxygen), because only polymers, which areprepared from hemoglobin that is deoxygenated during the cross-linkinghave the oxygen binding properties, which enable them to be used asartificial oxygen carriers for the desired indications. Preferably, asfurther protection against oxidation of the hemoglobin by traces ofremaining oxygen, the latter can be removed by chemical reaction withascorbate ions.

Especially pyridoxal-5′-phosphate is linked as effector of the oxygenbinding in an amount, optimized for the functional properties of the endproduct, covalently to the pig or human hemoglobin. This linking ofpyridoxal-5′-phosphate to hemoglobins basically is known; for example,patent EP-P 0 528 841 describes a method for the synthesis ofpyridoxylated hemoglobin. The pyridoxylation leads to the desiredhalf-saturation pressure value if the inventive procedure is employed.

The linking sites (aldimines=Schiffs bases) of the unstable covalentlinkage of pyridoxal-5′-phosphate can be stabilized, as is well known(see above), by reduction with sodium borohydride (amines are formed),the aforementioned special conditions being adhered to pursuant to theinvention. The capability of hemoglobin molecules for homotropiccooperativity of the oxygen binding sites with one another is generallyclearly lost by cross-linking the hemoglobin with the cross-linkingagent, glutardialdehyde. Pursuant to the invention, and to maintain thiscapability, 2,3-bisphosphoglycerate, a “heterotropic”, chemically notreactive effector of the oxygen binding of the hemoglobin, is addedbefore the cross-linking. Accordingly, this 2,3-bisphosphoglycerate canbe added reversibly to its binding site in the hemoglobin moleculeduring the cross-linking. After the synthesis, 2,3-bisphosphoglycerate,together with unused reactants as well as excess reaction product, isremoved completely (see below).

Glutardialdehyde is used as bifunctional cross-linking agent under theinventive conditions given above.

Two cross-linking sites (aldimines=Schiffs bases) of the cross-linkedmolecular glutardialdehyde bonds are stabilized, as described, byreduction with sodium borohydride (amines are formed), the inventiveconditions being observed.

Cross-linked hemoglobins are formed by the cross-linking and have amolecular weight distribution between about 50,000 and 5,000,000 g/mole(and larger, for example, 10,000,000 to 15,000,000 g/mole).

To improve the compatibility with plasma proteins, a polyethylene oxide(MW) derivative, especially the above-mentioned, monofunctional,activated (methoxy) polyethylene glycol having a molecular weight of1,500 to 2,500 g/mole, is linked to the cross-linked hemoglobin. Thelinking of polyethylene glycol (PEG), the so-called pegylation, to aswell as the cross-linking of hemoglobins is known (see also E. Tsucheda(Publisher); Blood Substitutes; Present and Future Perspectives,Elsevier Science, Amsterdam 1998).

However, the pegylation, as well as the cross-linking of hemoglobinstogether, as well as the use of the chemically inactive effector beforethe cross-linking, which contributes particularly to obtainingcooperativity as well as, in particular, the cross-linking conditionsdescribed, are new. The anyhow weak reaction of the RES and also theenzymatic degradation by proteases are prevented by the pegylationcarried out here.

The pH is adjusted as described above.

On the one hand, the development of a percolation distribution can beprevented by the inventive dilution and by the reaction sequence andconditions obtained. On the other hand, undesirable changes in theoxygen affinity and cooperativity can be avoided by the cross-linking ofand covalent attachment to hemoglobin by, for example, glutardialdehyde,pyridoxal phosphate and polyethylene oxide and, in particular, a highplasma compatibility can also be achieved.

All reaction steps together contribute to these special properties ofthe product produced pursuant to the invention.

The further working up is within the scope of knowledge of someoneskilled in the art. Insoluble components can be removed bycentrifugation (for example, for 20 minutes with a relative centrifugalacceleration of 20,000 g). The cross-linked hemoglobins are divided by a“well known” preparative step of the process, especially by a volumeexclusion chromatography or ultrafiltration, a factional precipitationor a field flow fractionation into a higher molecular weight and a lowermolecular weight fraction. If suitable methods are selected, (forexample, the nominal molecular-weight separation boundary of theultrafiltration membrane or the molecular weight separation range of thegel used are particularly important), the unused reactants as well asthe undesirable reaction products are removed at the same time.

A particularly preferred embodiment of the invention is explained ingreater detail by means of the following preparation example.

Purified pig or human hemoglobin, with a concentration between 200 and380 g/L and preferably between 240 and 360 g/L, is dissolved in anaqueous electrolyte. The latter contain sodium hydrogen carbonate at aconcentration between 10 and 150 mmoles/L and preferably between 40 and60 mmoles/L, and sodium chloride at a concentration of between 10 and150 moles/L and preferably between 50 and 100 mmoles per L. Thetemperature is 2° to 42° C. and preferably between 3° and 25° C. Thehemoglobin solution is stirred and pure nitrogen is passed over it inorder to deoxygenate the hemoglobin. To this solution, 2 to 8 moles andpreferably 3 to 4 moles of sodium ascorbate (as a 1 molar solution inwater) are added per mole of hemoglobin and allowed to react for aperiod between 0.5 and 6 hours and preferably between 70 and 120minutes.

The pH of the solution is now adjusted to a value between 6.5 and 7.5and preferably between 6.9 and 7.3 with lactic acid or sodium hydroxidesolution having a concentration between 0.1 and 1 and preferably between0.4 and 0.6 moles/L. Pyridoxal-5′-phosphate (0.5 to 3.0 and preferablyfrom 1.0 to 2.5 moles per mole of hemoglobin) is now added and allowedto react between 0.5 and 20 and preferably between 1 and 7 hours.

The pH is now adjusted with sodium hydroxide solution or lactic acid,having a concentration between 0.1 and 1 and preferably between 0.4 and0.6 moles/L to a value between 7.5 and 8.5 and preferably between 7.7and 8.2.

Now, 1.0 to 9.0 and preferably 1.0 to 2.5 moles of sodium borohydride(as a 1-molar solution in 0.01 molar sodium hydroxide solution) areadded and allowed to react for between 30 and 90 and preferably forbetween 50 and 70 minutes

The pH of the solution is adjusted with lactic acid or sodium hydroxidesolution, having a concentration between 0.1 and 1 and preferablybetween 0.4 and 0.6 moles/L) to a value between 6.5 and 7.5 andpreferably between 6.9 and 7.5

Now, 0.5 to 6.0 and preferably 1.0 to 4.0 moles of2,3-bisphosphoglycerate per mole of hemoglobin are added and allowed toreact for between 5 and 50 and preferably for between 10 and 20 minutes.

Subsequently, the time-controlled addition of the bifunctionalcross-linking agent is made. Between 6 and 10 and preferably between 7and 9 moles of glutardialdehyde per mole of hemoglobin, dissolved in 1to 4 and preferably 1.5 to 2 L of water per 1 of hemoglobin solution,are added within 3 to 10 and preferably within 4 to 6 minutes andallowed to react for between 1 and 6 and preferably for between 2 and 3hours.

The pH is adjusted with sodium hydroxide or lactic acid, having aconcentration between 0.1 and 1 and preferably between 0.4 and 0.6moles/L, to a value between 7.5 and 9.0 and preferably between 7.6 and8.8.

In all, 5 to 20 and preferably 6 to 12 moles of sodium borohydride (as a1-molar solution in 0.01 molar sodium hydroxide solution) is added permole of hemoglobin and allowed to react for between 15 and 100 andpreferably between 30 and 80 minutes. This is followed by the additionof 0 to 4 and preferably between 0.5 and 3 L of water per liter of theoriginal hemoglobin solution.

If necessary, the pH is adjusted with sodium hydroxide solution orlactic acid, having a concentration between 0.1 and 1 and preferablybetween 0.4 and 0.6 moles/L to a value between 7.5 and 10 and preferablybetween 8 and 9. Preferably, between 3 and 8 moles of an activatedpolyethylene oxide derivative, preferably methoxy-succinimidylpropionate polyethylene glycol, with a molecular weight between 500 and3000, preferably 1000 and 2500 and particularly 2000 g/mole are nowadded per mole of monomeric hemoglobin.

Subsequently, while continuing to stir the hemoglobin solution, thenitrogen atmosphere is replaced for 1 to 3 hours by pure oxygen and thehemoglobin thus is oxygenated swiftly. The working up is carried out asdescribed above.

The advantages of the inventive method can be summarized as follow.

Due to the inventive reaction sequence, especially the increase in thereaction volume in step iv) of the method, a product is obtained, whichcan be used completely for the preparation of artificial oxygencarriers. Moreover, about half of the product can be used as a bloodadditive (the fraction with the cross-linked hemoglobins of higherdegrees of polymerization, fraction I) and as blood volume substitute(fraction II), with the low molecular weight fractions. The separationcan be accomplished easily with known methods, some possible methodsbeing, for example, listed in the patents EP-A 95 107 280 and EP-A 97100 790. The polymers of fraction I, preferably up to a molecular weightof more than 700,000 g/mole, are so adequately uniform on a molecularbasis, that they have an adequately low colloidal osmotic pressure inthe desirably therapeutic plasma concentration. A small viralcoefficient as well as a low viscosity are achieved by this molecularuniformity. The comparability of the proteins of the blood plasma, thesufficiently large immune compatibility and intravasal residence time,as well as adequately low vasoconstrictive side effects, that is, a lowextravasation, of the polymers of fraction I is achieved by a covalentlinkage of the polyalkylene oxides. Moreover, the requirement ofsimplicity and economic efficiency is taken into account in a decisivemanner by this new method in that the whole preparation takes place in asingle vessel (a so-called one-vessel preparation) and in high yields ofmore than 70%, the yield of polymers with a molecular weight of morethan 700,000 g/mole exceeding 15%.

The method makes it possible to prepare modified and cross-linkedhemoglobins in a few process steps. The process parameters selected leadto a defined distribution of modified hemoglobin polymers, which, asartificial oxygen carriers, suitably take into account also thephysiological circumstances in the blood serum.

Moreover, the cooperativity of the uncrossed-link hemoglobin in thecross-linked product is largely obtained and the half saturationpressure can be adjusted suitably.

In parenteral application, the artificial oxygen carriers, producedpursuant to the invention from cross-linked hemoglobin, are compatiblewith the plasma and can be employed clinically as described.

The invention is described in greater detail by means of the followingexamples, FIGS. 1 to 3 showing the following:

FIG. 1 shows a mass average distribution of the molecular sizes andmolecular weights (M) of the pig hemoglobin polymers of Example 1, shownas a volume exclusion chromatogram, obtained with a Sephacryl S-400 HRgel, Pharmacia, Freiburg, Germany. E_(425 nm) is the extinction in thechromatography eluate. The abscissa values of 700,000 and 5,000,000g/mole are shown.

FIG. 2 shows a chromatogram for Example 2, the explanations being givenin FIG. 1.

FIG. 3 shows a chromatogram for Example 3, the explanations being givenin FIG. 1.

Furthermore, the following analytical methods were employed.

1. The hemoglobin contents were determined photometrically with themodified cyanhemoglobin method of Drabkin (“Hemoglobin Color Test MRP3”, Boehrimger Mannheim, Germany).

2. pH values were measured potentiometrically (glass electrode) with ablood-gas analyzer (“ABL 5”, Radiometer, Willich, Germany)

3. The molecular weight distribution of the cross-linked hemoglobins wasdetermined by volume-exclusion chromatography (Pötzschke H. et al.(1996): “Cross-linked Globular Proteins—A new class of semi-syntheticpolymeric molecules: characterization of their structure and solution insolution by means of volume-exclusion chromatography, viscosimetry,osmometry and light scattering using hyperpolymeric hemoglobins andmycoglobins as example”, Macromolecular Chemistry and Physics 197,1419-1437, as well as Pötzschke H. et al. (1996): “A novel method fordetermining molecular weights of widely distributed polymers with thehelp of gel photography and viscosimetry using hemoglobin hyperpolymersas examples”, Macromolecular Chemistry and Physics 197, 3229-3250) onSephacryl S-400 HR gel (Pharmacia Biotech, Freiburg, Germany).

4. The characteristics of oxygen binding by hemoglobins were determinedby means of our own methods and equipment (as described in Barnikol W.K. R. et al. (1978): “An improved modification of the Niesel and Thewsmicromethod for measuring O₂—Hb-binding curves in whole blood andconcentrated Hb solutions”, Respiration 36, 86-95).

5. The Plasma compatibility of cross-linked hemoglobins was investigatedby means of a standardized in vitro precipitation test (Domack, U.(1997), “Development and in vivo evaluation of an artificial oxygencarrier on the basis of bovine hemoglobin”, dissertation ChemistryDepartment of the Johannes Gutenberg University, Mainz 1997): Hemoglobinsolutions (with hemoglobin contents of about 30 g/L in an aqueouselectrolyte (StLg) containing 125 mM of sodium chloride, 4.5 mM ofpotassium chloride and 3 mM of sodium nitride) were mixed with equalamounts of freshly obtained, sterile filtered, human plasma.Subsequently, up to 20 μL of a 0.5 molar lactic acid was added to ineach case 500 μL of the mixture and mixed in, so that for eachhemoglobin derivative, which was to be investigated, the pH ranged from7.4 to 6.8. After an incubation period of 30 minutes at room temperatureand centrifugation of the samples, the hemoglobin content was determinedas a measure of the hemoglobin, which had not been precipitated, and theassociated pH in the supernatant was determined. In addition, asubjective, visual check for colorless precipitate of plasma proteinswas conducted.

EXAMPLE 1 Preparation of an Inventive Cross-Linked and MolecularlyModified Pig Hemoglobin at 4° C. by the General Method of Preparation

Pig hemoglobin of high purity, dissolved in a concentration of 330 g/Lin an aqueous electrolyte containing 50 mM of sodium bicarbonate and 100mM of sodium chloride, was deoxygenated at 4° C. by stirring thesolution under pure nitrogen, which was constantly being renewed.Subsequently, 4 moles of sodium ascorbate (as a I-molar solution inwater) was added per mole of (monomeric) hemoglobin and allowed to reactfor 6 hours. The pH of the solution was adjusted to a value of 7.1 with0.5-molar lactic acid. Subsequently, 1.1 mole of pyridoxal-5′-phosphatewere added per mole of hemoglobin and allowed to react for 16 hours. ThepH of the solution was now adjusted to a value of 7.8 with 0.5 molarsodium hydroxide solution and 1.1 moles of sodium borohydride (as a1-molar solution in 0.01-molar sodium hydroxide solution) were added andallowed to react for 1 hour. The pH was now adjusted to a value of 7.3with 0.5-molar lactic acid. Initially, 1.1 moles of2,3-bisphosphoglycerate per mole of hemoglobin and, after a reactionperiod of 15 minutes, 8 moles of glutardialdehyde per mole ofhemoglobin, dissolved in 1.8 L of pure water, was added per liter ofhemoglobin solution for cross-linking the hemoglobin within 5 minutesand allowed to react for 2.5 hours. After the pH was adjusted to a valueof 7.8 with a 0.5 molar sodium hydroxide solution, 15 moles of sodiumborohydride (as a 1-molar solution in 0.01 molar sodium hydroxidesolution) were added per mole of hemoglobin for 1 hour. Subsequently, 2L of water were added per liter of original hemoglobin solution. The pHthen was 9.3. Immediately thereafter, 4 moles of methoxysuccinimidylpropionate polyethylene glycol having a molecular weight of 2,000 g/molewere added over a period of 2 hours. The nitrogen atmosphere over thesolution was replaced by pure oxygen.

After 1 hour, the insoluble constituents were removed by centrifuging(20.000 g for 15 minutes). Subsequently, the electrolyte was changed bya volume exclusion chromatography (Sephadex G-25 gel, Pharmacia,Germany) to an aqueous electrolyte solution containing 125 mM of sodiumchloride, 4.5 mM of potassium chloride and 20 mM of sodium bicarbonate.

The yield was 77%; the yield for a molecular weight greater than 700,000g/mole is 28%.

FIG. 1 shows a representation of the distribution of the molar masses ofthe hemoglobin polymers obtained in the form of a volume exclusionchromatogram.

Measurements of the characteristic of the oxygen binding underphysiological conditions (a temperature of 37° C., a carbon dioxidepartial pressure of 40 torr and a pH of 7.4) reveal that the product hada p50 value of 22 torr and an n50 value of 1.95.

In the “precipitation test”, the cross-linked pig hemoglobin, in the pHrange of 7.4 to 6.8, showed no interactions whatsoever with humanplasma, especially no detectable precipitates of the hemoglobin or ofthe plasma proteins.

EXAMPLE 2 Preparation of an Inventive Cross-Linked and MolecularlyModified Pig Hemoglobin at Room Temperature by the General Method ofPreparation

Pig hemoglobin of high purity, dissolved in a concentration of 330 g/Lin an aqueous electrolyte containing 50 mM of sodium bicarbonate and 100mM of sodium chloride, was deoxygenated at 22° C. by stirring thesolution under pure nitrogen, which was constantly being renewed.Subsequently, 4 moles of sodium ascorbate (as a 1-molar solution inwater) was added per mole of (monomeric) hemoglobin and allowed to reactfor 90 minutes, the pH of the solution was adjusted to a value of 7.1with 0.5-molar lactic acid. Subsequently, 1.1 moles ofpyridoxal-5′-phosphate were added per mole of hemoglobin and allowed toreact for 2 hours. The pH of the solution was now adjusted to a value of7.8 with 0.5 molar sodium hydroxide solution and 1.5 moles of sodiumborohydride (as a 1-molar solution in 0.01-molar sodium hydroxidesolution) were added and allowed to react for 1 hour. The pH was nowadjusted to a value of 7.3 with 0.5-molar lactic acid. Initially, 1.5moles of 2,3-bisphosphoglycerate per mole of hemoglobin and, after areaction period of 15 minutes, 9 moles of glutardialdehyde per mole ofhemoglobin, dissolved in 1.8 L of pure water, was added per liter ofhemoglobin solution within 5 minutes, for cross-linking the hemoglobinand allowed to react for 1 hour. After the pH was adjusted to a value of7.8 with a 0.5 molar sodium hydroxide solution, 10 moles of sodiumborohydride (as a 1-molar solution in 0.01 molar sodium hydroxidesolution) were added per mole of hemoglobin for 30 minutes. The pH was8.7. Immediately thereafter, 8 moles of methoxysuccinimidyl propionatepolyethylene glycol having a molecular weight of 1000 g/mole, were addedover a period of 1 hour. The nitrogen atmosphere over the solution wasreplaced with pure oxygen.

After 1 hour, the insoluble constituents were removed by centrifuging(20.000 g for 10 minutes). Subsequently, the electrolyte was changed byvolume exclusion chromatography (Sephadex G-25 gel, Pharmacia, Germany)to an aqueous electrolyte solution containing 125 mM of sodium chloride,4.5 mM of potassium chloride and 20 mM of sodium bicarbonate.

The yield was 79%; the yield for a molecular weight greater than 700,000μmole was 28%.

FIG. 2 shows a representation of the distribution of the molar masses ofthe hemoglobin polymers obtained in the form of a volume exclusionchromatogram.

Measurements of the characteristic of the oxygen binding underphysiological conditions (a temperature of 37° C., a carbon dioxidepartial pressure of 40 torr and a pH of 7.4) reveal that the product hada p50 value of 22 torr and an n50 value of 1.6.

In the “precipitation test”, the cross-linked pig hemoglobin, in thephysiologically and pathophysiologically interesting pH range of 7.4 to6.8, showed no interactions whatsoever with human plasma, especially nodetectible precipitates of the hemoglobin or the plasma proteins.

EXAMPLE 3 Preparation of an Inventive Cross-Linked and MolecularlyModified Human Hemoglobin at 4° C. by the General Method of Preparation

Human hemoglobin of high purity, dissolved in a concentration of 330 g/Lin an aqueous electrolyte containing 50 mM of sodium bicarbonate and 100mM of sodium chloride, was deoxygenated at 4° C. by stirring thesolution under pure nitrogen, which was constantly being renewed.Subsequently, 4 moles of sodium ascorbate (as a I-molar solution inwater) was added per mole of (monomeric) hemoglobin and allowed to reactfor 3 hours. The pH of the solution was adjusted to a value of 7.1 with0.5-molar lactic acid. Subsequently, 1.1 mole of pyridoxal-5′-phosphatewere added per mole of hemoglobin and allowed to react for 16 hours. ThepH of the solution was now adjusted to a value of 7.8 with 0.5 molarsodium hydroxide solution and 1.1 moles of sodium borohydride (as a1-molar solution in 0.01-molar sodium hydroxide solution) were added andallowed to react for 1 hour. The pH was now adjusted to a value of 7.3with 0.5-molar lactic acid. Initially, 1.5 moles of2,3-bisphosphoglycerate per mole of hemoglobin and, after a reactionperiod of 15 minutes, 9 moles of glutardialdehyde per mole ofhemoglobin, dissolved in 1.8 L of pure water, were added uniformlywithin 5 minutes per liter of hemoglobin solution for cross-linking thehemoglobin and allowed to react for 2.5 hours. After the pH was adjustedto a value of 8.0 with a 0.5 molar sodium hydroxide solution, 10 molesof sodium borohydride (as a 1-molar solution in 0.01 molar sodiumhydroxide solution) were added per mole of hemoglobin for 1 hour.Subsequently, 2 L of water were added per liter of original hemoglobinsolution. The pH then was 8.6. Immediately thereafter, 4 moles ofmethoxysuccinimidyl propionate polyethylene glycol, having a molecularweight of 2000 g/mole, was added over a period of 2 hours. The nitrogenatmosphere over the solution was replaced with pure oxygen.

After 1 hour, the insoluble constituents were removed by centrifuging(20.000 g for 10 minutes). Subsequently, the electrolyte was changed bya volume exclusion chromatography (Sephadex G-25 gel, Pharmacia,Germany) to an aqueous electrolyte solution containing 125 mM of sodiumchloride, 4.5 mM of potassium chloride and 20 mM of sodium bicarbonate.

FIG. 3 shows a representation of the distribution of the molar masses ofthe hemoglobin polymers obtained in the form of a volume exclusionchromatogram.

The total yield was 75%; the yield of polymers with a molecular weightgreater than 700,000 g/mole was 17%.

Measurements of the characteristic of the oxygen binding underphysiological conditions (a temperature of 37° C., a carbon dioxidepartial pressure of 40 torr and a pH of 7.4 reveal that the product hada p50 value (as a measure of the average oxygen affinity) of 21 torr andan n50 value (an average apparent cooperativity of the oxygen bindingsites) of 1.74.

In the “precipitation test”, the cross-linked pig hemoglobin, in thephysiologically and pathophysiologically interesting pH range of 7.4 to6.8, showed no interactions whatsoever with human plasma, especially nodetectible precipitates of the hemoglobin or of plasma proteins.

1. Method for the production of artificial oxygen carriers comprisingcrosslinked hemoglobin, said method comprising the following steps: i)deoxygenizing a first solution comprising hemoglobin to form a secondsolution comprising deoxygenated hemoglobin; ii) covalently reacting thesecond solution comprising deoxygenated hemoglobin with a chemicallyreactive effector of an oxygen bonding to form a third solution; iii)treating the third solution with a non-chemically reactive effector ofan oxygen bonding to form a fourth solution; and then iv) stablycovalently crosslinking the hemoglobin with glutaraldehyde by dilutingthe fourth solution and adding a crosslinking agent, then furtherdiluting the fourth solution, while the volume of the fourth solutionincreases overall by a factor of 2 to 10; and then v) covalently linkinga polyethylene oxide derivative to the hemoglobin; and vi) processingand formulating a resulting product as a pharmaceutical.
 2. Methodaccording to claim 1, wherein the hemoglobin originates from pigs orhuman beings.
 3. Method according to claim 2, wherein the hemoglobinoriginates from pigs.
 4. Method according to claim 1, wherein in stepiv) glutaraldehyde is added to the hemoglobin solution while dilutingthe hemoglobin solution up to 5- to 6-fold under time control.
 5. Methodaccording to claim 4, wherein in step iv) the hemoglobin is monomerichemoglobin, and glutaraldehyde is added within 3-15 minutes in an amountof 6-10 mol, with respect to the monomeric hemoglobin, dissolved in 1-2liters of water per liter of original reaction solution, and reacted foranother 1-6 hours.
 6. Method according to claim 1, wherein to the firstsolution comprising the hemoglobin, before step ii), there is added 2 to8 mol sodium ascorbate per mol of uncrosslinked hemoglobin for 0.5-6hours.
 7. Method according to claim 1, wherein in step ii) thehemoglobin is monomeric hemoglobin, the chemically reactive effector ofan oxygen bonding is pyridoxal-5′-phosphate, and thepyridoxal-5′-phosphate is covalently bonded onto the monomerichemoglobin in a molar ratio, with respect to the monomeric hemoglobin,of 0.5 to 3 mol/mol within 0.5 to 20 hours.
 8. Method according to claim7, wherein in steps ii) and iv), after the covalent bonding ofpyridoxal-5′-phosphate onto the monomeric hemoglobin, and after covalentcrosslinking of the monomeric hemoglobin with glutaraldehyde, reductivesodium borohydride is added in each case.
 9. Method according to claim1, wherein in step iii) the hemoglobin is monomeric hemoglobin, thenon-chemically reactive effector of an oxygen bonding is2,3-bisphosphoglycerate, and the 2,3-bisphosphoglycerate is added in arelative amount of 0.5-6 mol with respect to the monomeric hemoglobin,and step iv) is started 5 to 50 minutes thereafter.
 10. Method accordingto claim 1, wherein in step v), the polyethylene oxide derivative is apolyethylene glycol ester with a molecular weight of 500 to 3000 g/mol.11. Method according to claim 1, wherein the product obtained isseparated by a preparative separating process into a fraction of greateraverage molecular mass and a fraction of lower average molecular mass.12. Method according to claim 11, wherein a parenteral blood substituteis prepared from the low molecular weight fraction and a parenteralblood additive is prepared from the higher molecular weight fraction.13. Artificial oxygen carrier which is a hemoglobin crosslinked andpolymerized with glutaraldehyde and covalently linked with apolyethylene oxide derivative.
 14. Artificial oxygen carrier accordingto claim 13, wherein the carrier has an n50 value of 1.6 to 2.5 and ap50 value of 16 to 24 Torr.
 15. Method of using an artificial oxygencarrier according to claim 13 for intravascular or biomedicalapplication as a synthetic oxygen carrier, said method comprisingadministering said artificial oxygen carrier to a patient in needthereof.
 16. Method according to claim 15, which comprises administeringthe artificial oxygen carrier to the patient in need thereof as asubstitute for blood (blood substitute) or as an additive to the bloodto increase the oxygen transport capacity (blood additive) or in anutrient solution.
 17. Method according to claim 16, which is carriedout for the treatment of chronic oxygen deficiency in a human being. 18.Method according to claim 7, wherein in step ii) the hemoglobin ismonomeric hemoglobin, the chemically reactive effector of an oxygenbonding is pyridoxal-5′-phosphate, and the pyridoxal-5′-phosphate iscovalently bonded onto the monomeric hemoglobin in a molar ratio, withrespect to the monomeric hemoglobin, of 1 to 2.5 mol/mol within 0.5 to20 hours.